The application addresses the broad Challenge Area (15) Translational Science and Specific Challenge Topic, 15-ES-101;Effects of environmental exposures on phenotypic outcomes using non-human models. This application will apply non-human modeling to examine a genetic mutation which may be responsible for increasing susceptibility to develop manganese (Mn) toxicity. Chronic exposure to the Mn has been linked to development of a severe, irreversible neurological disorder known as manganism consisting of reduced response speed, intellectual deficits and mood changes in the initial stages of the disorder to more prominent and irreversible extrapyramidal dysfunction resembling Parkinson's disease upon protracted exposure. Manganism is primarily an occupational disorder associated with environmental conditions in which workers are exposed to chronic high levels of the metal and include Mn miners, welders and those involved in ferroalloy processing. The CNS injury caused by Mn manifests in a diverse set of symptoms indicative of the fact that the basal ganglia is one of the most complex areas of the brain. When in excess, Mn disrupt the delicate balance between the well organized and interrelated components within the basal ganglia as a disturbance in any one of the elements, whether it occurs in the globus pallidus as in manganism or the nigro-striatal neurons as in Parkinson's disease, manifests in insidious neurological deficits that, in some respect, can appear remarkably similar. Differences in response to Mn overexposure in the human population, most likely, are due to underlying genetic variability which ultimately presents in deviations in both susceptibility as well as the characteristics of the neurological lesions and symptoms expressed. Although the underlying genetic defect responsible for increased susceptibility to develop manganism is not known, our preliminary findings revealed that the gene for early onset of Parkinson's disease, parkin, has the potential to regulate Mn toxicity and, thus for the first time, identify a potential genetic link between manganism and Parkinsonism. Parkin is an E3 ligase responsible for ubiquitinating a number of proteins destined for degradation via the proteasomal pathway. Of the known Parkinson's disease -linked genetic mutations correlating with early onset, those involving parkin are the most prevalent encompassing approximately 50% of all recessive Parkinson's disease cases. Our preliminary studies have now established (unpublished findings) that parkin is the E3 ligase responsible for the ubiquitination of the 1B isoforms of divalent metal transporter 1 (DMT1), the major protein for transport of Mn. These studies reveal that neuroblastoma cells overexpressing parkin display both decreased expression of DMT1 as well as increased toxicity towards Mn. Non-transformed human lymphocytes that are homozygous for the mutation in exon 4 of parkin express increased levels of the transporter as do brains from parkin knock-out mice. Although considered to be an autosomal recessive gene there is evidence in the literature suggesting that mutations in a single allele of the parkin gene may exert sufficient imbalance in dopaminergic activity to cause subclinical features of Parkinsonism and play a significant role in idiopathic Parkinson's disease. Thus, individuals that are heterozygous for the parkin mutation may similarly be at greater risk to develop Mn toxicity. In addition to the ability of parkin to alter DMT1 expression, mutations in the gene have also been reported to regulate activation of a number of signaling processes associated with Mn toxicity. The contribution of these parkin-induced changes in response to oxidative and toxic insults and their subsequent role in stimulating Mn toxicity is not known but we hypothesize they may likely accelerate the biochemical systems initiating cell death. The major mechanism contributing to Mn toxicity is via disruption of mitochondrial function and the subsequent activation of oxidative stress-related processes which ultimate manifests in changes in classical markers and signaling pathways associated with apoptotic cell death. Accordingly, the experiments proposed in this grant application will address this issue as we propose to determine the influence that mutations in the parkin gene have on cell signaling as it relates to Mn toxicity in non-human models. Changes in known Mn-inducing apoptotic and mitochondrial signaling processes will be monitored in the SH-SY5Y human neuroblastoma cells transfected with vector only, wild-type and mutant constructs of the parkin gene. For the knock-down model, we will use three human lymphocyte cell lines (wt and heterozygous and homozygous for the parkin mutation) as application of these latter cells eliminates any problems that may be associated with transfection effects in the SH-SY5Y cells and assures that we have totally abolished parkin activity. In addition, we will compare signaling processes in brains of control and parkin knockout mice treated with Mn. The litany of assays described in this application using non- human models is designed to obtain a comprehensive overview of the changes in the Mn-stimulated pathways that may be altered by mutations in the parkin gene. PUBLIC HEALTH RELEVANCE: Overexposure to high atmospheric levels of manganese leads to a syndrome known as manganism which is characterized by an irreversible extrapyramidal dysfunction resembling that of Parkinson's disease. Our preliminary studies, for the first time, reveal a link between manganism and Parkinson's disease in that the gene evoking early onset of Parkinsonism, parkin, can prevent Mn toxicity. Thus, the studies proposed in this grant are relevant to human health in that they will investigate the mechanism by which mutations in the parkin gene regulate and increase vulnerability to develop manganism.